Ultra turrax t8 homogenizer

All animal experiments were approved by the Austrian Ministry of Science (BMWF-66.011/0075- WF/V/3b/2016) and performed in accordance with the Austrian Animal Protection Act. Metabolic studies characterizing the stability of selected radioligands against enzymatic degradation were carried out in 7 to 8 week old female BALB/c mice (Charles River, Sulzfeld, Germany). The mice (two animals for each peptide) were intravenously injected into a lateral tail vein with 5–10 MBq of the ¹¹¹In-labeled peptide derivatives, corresponding to 2 nmol total peptide. At the time point of 10 min p.i. the animals were euthanatized by cervical dislocation. A sample of urine and blood was immediately collected. Liver and kidneys were dissected, mixed with 20 mM HEPES buffer pH 7.3 (1:1, w/v) and homogenized using an Ultra-Turrax T8 homogenizer (IKA-Werke, Staufen, Germany) for 1 min. All samples taken were analyzed by analytical HPLC using the low-sensitivity loop for urine and the high-sensitivity loop for blood, liver and kidney. Before injection into the HPLC system, all samples except urine were treated with ACN and diluted with water as described for stability studies in human serum. The percentage of intact radiopeptide in the analyzed samples was calculated by integration of the different peaks found in the radiochromatogram using Chromeleon Dionex Software (Version 7.2.9.11323).

Pieces weighing between 80 and 320 mg were homogenized in lysis buffer (15 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 0.5 % Triton X-100), with addition of protease inhibitor (Thermo Scientific, Waltham, MA, USA) using an IKA Ultra-Turrax T8 homogenizer (IKA-Werke, Staufen im Breisgau, Germany). Homogenates were centrifuged at 300×g at 4 °C for 10 minutes. Supernatants were kept at −80 °C until assay. Cytokine content was assessed using a DuoSet enzyme-linked immunosorbent assay (ELISA) system (R&D Systems, Minneapolis, MN, USA), for porcine tumor necrosis factor (TNF)-α, interleukin (IL)-1β/IL-1F2, and IL-6 according to the manufacturer’s instructions. The detection limits of the assays were 125 pg/ml for TNF-α, 62.5 pg/ml for IL-1β/IL-1F2, and 125 pg/ml for IL-6. Total protein content of the supernatant was measured using a Coomassie Plus Assay (Thermo Scientific) according to the manufacturer’s instructions. Cytokine content of tissue lysates were normalized against total protein content of the homogenate.

The brain tissues were thawed on ice and homogenized using Ultra-turrax T8 homogenizer (IKA-Werke GmbH & Co. KG, Germany) in solubilization buffer containing 8M urea, 2% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 0.2% sodium orthovanadate and 1 × concentration of protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). The supernatant was collected by centrifugation and subjected to three pulses of sonication on ice followed by centrifugation at 20,000 × g for 30 min at 4 °C for the recovery of total soluble proteins. Protein extraction from Neuro2a cells was carried out in a similar manner. The rest of the extraction procedure was done according to ref. 51 (link).

Metabolic and biodistribution studies were performed in accordance with the ethical standards of the institution and approved by the Austrian Ministry of Science (BMWFW-66.011/0072-V/3b/2019).
In vivo stability studies of [¹⁷⁷Lu]Lu-1 and [¹⁷⁷Lu]Lu-2 were carried out in 5–7-week-old female BALB/c mice (Charles River Laboratories, Sulzfeld, Germany; n = 6). A higher radioactivity of ~37 MBq in a total volume of ~150 µL in PBS/0.5% BSA, corresponding to ~1 nmol total peptide, was administered into the mice intravenously through a lateral tail vein to increase the detectability of potential radiometabolites by radio-HPLC. The mice were euthanized by cervical dislocation after 10 (n = 2) and 30 (n = 1) min post injection and the urine and a venous blood sample were collected at the time of sacrifice. Liver and kidneys were dissected and homogenized in 20 mM HEPES buffer pH 7.3 (1:1, v/v) with an Ultra-Turrax T8 homogenizer (IKA-Werke, Staufen, Germany) for 1 min at RT. Prior to the radio-HPLC analysis, the samples of blood, kidney, and liver homogenates were treated with ACN to precipitate proteins (1:1, v/v), centrifuged (2000× g, 2 min) and the supernatant was diluted with water (1:1, v/v). Urine was diluted 1:4 with water before injection.

We used skeletal muscle which were isolated for previously published study (See extended methods Schönke et al.). Frozen gastrocnemius muscles were crushed using mortar and pestle. Powdered muscle was homogenized using Ultra Turrax T8 homogenizer (IKA Labortechnik, Staufen im Breisgau, Germany) in 4% SDS buffer (100 mm Tris-HCl, pH 7.4). Protein lysates were boiled at 100 °C for 5 mins. Lysates were sonicated using a tip and centrifuged at 16,000 × g for 10 mins followed by reduction and alkylation as described above, the supernatant was then processed using FASP or PAC or frozen until further analysis.